Process for preparing an A2A-adenosine receptor agonist and its polymorphs

ABSTRACT

Disclosed is a synthesis suitable for large scale manufacture of an A 2A -adenosine receptor agonist, and also relates to polymorphs of that compound, and to methods of isolating a specific polymorph.

This application is a continuation of U.S. patent application Ser. No.12/687,077, filed Jan. 13, 2010, now U.S. Pat. No. 7,956,179, whichclaims priority to U.S. patent application Ser. No. 11/750,295, filedMay 17, 2007, now U.S. Pat. No. 7,671,192, which is acontinuation-in-part of U.S. patent application Ser. No. 11/701,699,filed Feb. 2, 2007, now U.S. Pat. No. 7,732,595, which claims priorityto U.S. Provisional Patent Application Ser. No. 60/801,857, filed May18, 2006, and to U.S. Provisional Patent Application Ser. No.60/765,114, filed Feb. 3, 2006, which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a process for the large scalepreparation of an A_(2A)-adenosine receptor agonist, and also relates topolymorphs of that compound, and to methods of isolating a specificpolymorph.

BACKGROUND

Adenosine is a naturally occurring nucleoside, which exerts itsbiological effects by interacting with a family of adenosine receptorsknown as A₁, A_(2A), A_(2B), and A₃, all of which modulate importantphysiological processes. One of the biological effects of adenosine isto act as a coronary vasodilator; this result being produced byinteraction with the A_(2A) adenosine receptor. This effect of adenosinehas been found to be useful as an aid to imaging of the heart, wherecoronary arteries are dilated prior to administration of an imagingagent (for example, thallium 201), and thus, by observation of theimages thus produced, the presence or absence of coronary artery diseasecan be determined. The advantage of such a technique is that it avoidsthe more traditional method of inducing coronary vasodilation byexercise on a treadmill, which is clearly undesirable for a patient thathas a coronary disease.

However, administration of adenosine has several disadvantages.Adenosine has a very short half life in humans (less than 10 seconds),and also has all of the effects associated with A₁, A_(2A), A_(2B), andA₃ receptor agonism. Thus the use of a selective A_(2A) adenosinereceptor agonist would provide a superior method of producing coronaryvasodilation, particularly one with a longer half life and few or noside effects.

A class of compounds possessing these desirable properties was disclosedin U.S. Pat. No. 6,403,567, the complete disclosure of which is herebyincorporated by reference. In particular, one compound disclosed in thispatent,(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamide,has been shown to be a highly selective A_(2A)-adenosine receptoragonist, and is presently undergoing clinical trials as a coronaryvasodilator useful in cardiac imaging.

Given the heightened interest in this and similar compounds, it hasbecome desirable to find new methods of synthesis that provide aconvenient method for making large quantities of the material in goodyield and high purity. The patent that discloses the compound ofinterest (U.S. Pat. No. 6,403,567) provides several methods forpreparing the compound. However, although these methods are suited tosmall scale syntheses, all synthetic methods disclosed in the patentutilize protecting groups, which is undesirable for large scalesyntheses.

Additionally, it was discovered that the desired product (that is(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamide)is capable of existing in at least three different crystalline forms,the most stable of which is a monohydrate. This polymorph is stableunder relative humidity stress conditions, up to its melting point.Accordingly, it is desirable that the final product produced in the newsyntheses is obtained as the stable monohydrate.

SUMMARY OF THE INVENTION

Thus, it is an object of this invention to provide convenient synthesesfor the large scale preparation of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamide,and polymorphs thereof, preferably as its monohydrate. Accordingly, in afirst aspect, the invention relates to the preparation of a compound ofthe Formula I:

comprising:contacting a compound of the formula (3):

with methylamine.

In one embodiment the reaction is conducted in an aqueous solution ofmethylamine, initially at a temperature of about 0-5° C., followed bywarming to about 50-70° C. Alternatively, the reaction is conducted asabove but in a sealed pressure reactor.

In a second embodiment, the product is isolated as the pure monohydrateby dissolving the product in a solvent, for example dimethylsulfoxide,addition of purified water, filtering the slurry thus formed, washingthe contents of the filter with water followed by ethanol, and dryingthe solid that remains under vacuum at a temperature that does notexceed 40° C.

In a second aspect, the invention relates to the preparation of acompound of the formula (3):

comprising:contacting a compound of the formula (2):

with ethyl 2-formyl-3-oxopropionate.

In one embodiment, the reaction is conducted in ethanol, at atemperature of about 80° C., with about 1.1 molar equivalents of ethyl2-formyl-3-oxopropionate.

In a third aspect, the invention relates to the preparation of acompound of the formula (2):

comprising:contacting a compound of the formula (1):

with hydrazine.

A 14.3-16.7 fold molar excess of hydrazine may be used and the reactionmay be conducted at a temperature of approximately 60-65° C. In oneembodiment the hydrazine is first heated to approximately 60-65° C.followed by addition of the compound of formula (1). The compound offormula (2) may be isolated by (a) cooling the reaction mixture toapproximately 40° C., (b) adding a 4.2-4.9 mass equivalent of waterwhile maintaining the temperature at approximately 40° C., (c) coolingthe mixture to approximately 10° C. and maintaining at that temperaturefor at least approximately 1 hour, (d) filtering, (e) washing thecontents of the filter with water followed by ethanol, and (f) dryingthe solid that remains under vacuum at a temperature that does notexceed 30° C. for at least 12 hours.

The above described synthesis is suitable for the large scale synthesisof the desired product, which is provided in good yield, although oneminor impurity is seen in the final product. This impurity has beenshown to be unchanged intermediate of the formula (2); that is, thecompound of the formula:

Although this impurity can be removed from the final product bycrystallization, it was decided to seek an alternative synthesis thathad all of the advantages of the above synthesis but did not give thecompound of formula (2) as an impurity in the final product.

Thus, in a fourth aspect, the invention relates to a method ofsynthesizing(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideby contacting a compound of the formula (4):

with methylamine.

In one embodiment the reaction is conducted in an aqueous solution ofmethylamine, initially at a temperature of about 0-5° C., followed bywarming to about 50-70° C. Preferably, the reaction is conducted in asealed pressure reactor.

In another embodiment, the reaction is conducted at a temperature ofapproximately 2.5-7.5° C.

In still another embodiment, the product is isolated as the puremonohydrate by dissolving the product in a solvent, for exampledimethylsulfoxide, addition of purified water, filtering the slurry thusformed, washing the contents of the filter with water followed byethanol, and drying the solid that remains under vacuum at a temperaturethat does not exceed 40° C. In some other embodiments, the product maybe isolated as the pure monohydrate by (a) degassing the reactionmixture under vacuum at not more than 35° C. to remove excessmethylamine, (b) releasing the vacuum and cooling to 0-5° C. forapproximately 15-minutes to an hour, (c) filtering the slurry thusformed, (d) washing the contents of the filter with water followed byethanol, and (e) drying the solid that remains under vacuum at atemperature that does not exceed 40° C.

In some embodiments the(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideis further purified to provide the monohydrate form by (i) dissolvingthe compound in a solvent, such as dimethylsulfoxide, heated toapproximately 78-88°, (ii) filtering any solid impurities from thesolution, (iii) rinsing with additional solvent, (iv) adding solution topurified water that is maintained at approximately 78-88° therebyforming a slurry, (v) stirring the slurry, (vi) cooling the slurry,(vii) filtering, (viii) washing the contents of the filter with waterfollowed by ethanol, and (ix) drying the solid that remains under vacuumat a temperature that does not exceed 40° C.

In a fifth aspect, the invention relates to a method of synthesizing acompound of the formula (4):

comprising contacting a compound of the formula (2):

with an excess of ethyl 2-formyl-3-oxopropionate, preferably about a2-10 fold molar excess, more preferably about a 5-10 fold excess. In oneembodiment, the reaction is conducted in ethanol, at a temperature ofabout 80° C. The ethyl 2-formyl-3-oxopropionate is present in a 5-10fold excess.

In a further embodiment, an additional method of synthesizing a compoundof the formula (4) is also presented comprising contacting a compound ofthe formula (2)

with an excess of ethyl 2-formyl-3-oxopropionate in the presence ofacid. The reaction takes place at reflux is generally conducted inethanol. The reaction takes place with or without the use of HCl as acatalyst. Up to 0.1 molar equivalent, preferably about a 0.05 molarexcess of HCl and about a 5-10 fold molar excess, preferably about a6.8-7.5 fold molar excess, of ethyl 2-formyl-3-oxoproionate are used.The product of this reaction may be isolated by (a) cooling thecompleted reaction mixture to approximately 10° C., (b) filtering, (c)washing the contents of the filter with ethanol, and (d) drying thesolid that remains under vacuum at a temperature that does not exceed40° C.

DEFINITIONS AND GENERAL PARAMETERS

FIG. 1 is a ¹H NMR spectrum of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidemonohydrate (Form A).

FIG. 2 shows the thermal analysis of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidemonohydrate.

FIG. 3 shows the X-Ray diffraction pattern for(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethypoxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidemonohydrate.

FIG. 4 shows the X-Ray diffraction pattern for(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideForm B.

FIG. 5 shows the X-Ray diffraction pattern for(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideForm C as compared to Form A.

As used in the present specification, the following words and phrasesare generally intended to have the meanings as set forth below, exceptto the extent that the context in which they are used indicatesotherwise.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not.

The term “therapeutically effective amount” refers to that amount of acompound of Formula I that is sufficient to effect treatment, as definedbelow, when administered to a mammal in need of such treatment. Thetherapeutically effective amount will vary depending upon the subjectand disease condition being treated, the weight and age of the subject,the severity of the disease condition, the manner of administration andthe like, which can readily be determined by one of ordinary skill inthe art.

The term “treatment” or “treating” means any treatment of a disease in amammal, including:

-   -   (i) preventing the disease, that is, causing the clinical        symptoms of the disease not to develop;    -   (ii) inhibiting the disease, that is, arresting the development        of clinical symptoms; and/or    -   (iii) relieving the disease, that is, causing the regression of        clinical symptoms.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The term “polymorph” is intended to include amorphous and solvates of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamide.

It has been discovered that this compound is capable of existing in atleast three different crystalline forms, referred to herein as Form A,Form B, Form C, and an amorphous product.

Form A: This polymorph can be produced by crystallizing1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidefrom protic solvents, for example ethanol or ethanol/water mixtures, orfrom a polar solvent, for example dimethylsulfoxide/water. Form A hasbeen shown to be a monohydrate, and is the most stable of the variouspolymorphs at ambient temperatures. It is stable under relative humiditystress conditions up to its melting point.

Form B: This polymorph is produced by evaporating under vacuum asolution of1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidein trifluoroethanol at ambient temperatures. The X-ray analysis of thecrystals was distinctly different from any other polymorph (see FIG. 4),but it was difficult to determine its constitution, as the X-rayanalysis gave disordered broad peaks, and the polymorph containedvarying amounts of water. It was found to be difficult to reliablyreproduce the preparation of this polymorph.

Form C: This polymorph is produced by slurrying1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidein acetonitrile for a long period of time at 60° C. The X-ray analysisof the crystals was distinctly different from any other polymorph (seeFIG. 5). Polymorph C was shown to be a variable hydrate, which, atelevated temperatures, desolvates to an unstable form.

Amorphous Material: This polymorph is produced by heating Form Apolymorph at a temperature of up to 200° C. This polymorph is unstablein the presence of atmospheric moisture, forming variable hydrates.

Techniques for Analysis of Forms A, B, C and Amorphous Material

X-Ray Powder Diffraction

X-ray powder diffraction (XRPD) analyses were carried out on a ShimadzuXRD-6000 X-ray powder diffractometer using Cu Kα radiation. Theinstrument was equipped with a fine focus X-ray tube, and the tubevoltage and amperage were set to 40 kV and 40 mA respectively. Thedivergence and scattering slits were set at 1″ and the receiving slitwas set at 0.15 mm. Diffracted radiation was detected by a NaIscintillation detector. A theta-two theta continuous scan at 3°/min (0.4sec/0.02° step) from 2.5-40° 2θ was used. A silicon standard was used tocheck the instrument alignment. Data were collected and analyzed usingXRD-6000 v. 4.1 software.

X-ray powder diffraction (XRPD) analyses were also performed using anInel XRG-3000 diffractometer equipped with a CPS (Curved PositionSensitive) detector with a 28 range of 120°. The instrument calibrationwas performed using a silicon reference standard. The tube voltage andamperage were set to 40 kV and 30 mA, respectively. The monochromatorslit was set at 5 mm by 80 μm. Samples were placed in an aluminum sampleholder with a silicon insert or in glass XRPD-quality capillaries. Eachcapillary was mounted onto a goniometer head that is motorized to permitspinning of the capillary during data acquisition. Real time data werecollected using Cu-Kα radiation at a resolution of 0.03° 2θ. Typically,data were collected over a period of 300 seconds. Only the data pointswithin the range of 2.5-40° 2θ are displayed in the plotted XRPDpatterns.

Thermal Analyses

Thermogravimetric (TG) analyses were carried out on a TA Instruments2050 or 2950 thermogravimetric analyzer. The calibration standards werenickel and Alumel™. Samples were placed in an aluminum sample pan,inserted into the TG furnace, and accurately weighed. The samples wereheated in nitrogen at a rate of 10° C./min to either 300 or 350° C.Unless stated otherwise, sample weights were equilibrated at 25° C. inthe TGA furnace prior to analysis.

Differential scanning calorimetry (DSC) analyses were carried out on aTA Instruments differential scanning calorimeter 2920. Accuratelyweighed samples were placed in either crimped pans or hermeticallysealed pans that contained a pinhole to allow for pressure release. Eachsample was heated under nitrogen at a rate of 10° C./min to either 300or 350° C. Indium metal was used as the calibration standard.Temperatures were reported at the transition maxima.

Infrared Spectroscopy

Infrared spectra were acquired on Magna 860® Fourier transform infrared(FT-IR) spectrophotometer (Nicolet Instrument Corp.) equipped with anEver-Glo mid/far IR source, an extended range potassium bromidebeamsplitter, and a deuterated triglycine sulfate (DTGS) detector.Unless stated otherwise, a Spectra-Tech, Inc. diffuse reflectanceaccessory (the Collector™) was used for sampling. Each spectrumrepresents 256 co-added scans at a spectral resolution of 4 cm⁻¹. Samplepreparation for the compound consisted of placing the sample into amicrocap and leveling the material with a frosted glass slide. Abackground data set was acquired with an alignment mirror in place. Thespectra represent a ratio of the sample single-beam data set to thebackground single beam data set. Wavelength calibration of theinstrument was performed using polystyrene.

NMR Spectroscopy

Solution phase ¹H NMR spectra of the were acquired at ambienttemperature on a Bruker model AM-250 spectrometer operating at 5.87 T(Larmor frequency: ¹H=250 MHz). Time-domain data were acquired using apulse width of 7.5 ps and an acquisition time of 1.6834 second over aspectral window of 5000 Hz. A total of 16,384 data points werecollected. A relaxation delay time of 5 seconds was employed betweentransients. Each data set typically consisted of 128 coaveragedtransients. The spectra were processed utilizing GRAMS132 A1 software,version 6.00. The free induction decay (FID) was zero-filled to fourtimes the number of data points and exponentially multiplied with aline-broadening factor of 0.61 Hz prior to Fourier transformation. The¹H spectra were internally referenced to tetramethylsilane (0 ppm) thatwas added as an internal standard.

Alternatively, NMR analysis was carried out as described in Example 4.

Moisture Sorption/Desorption Analyses

Moisture sorption/desorption data were collected on a VTI SGA-100 VaporSorption Analyzer. Sorption and desorption data were collected over arange of 5% to 95% relative humidity (RK) at 10% RH intervals under anitrogen purge. Sodium chloride (NaCl) and polyvinylpyrrolidone (PVP)were used as the calibration standards. Equilibrium criteria used foranalysis were less than 0.0100% weight change in 5 minutes, with amaximum equilibration time of 180 minutes if the weight criterion wasnot met. The plotted data have not been corrected for the initialmoisture content.

Nomenclature

The structure of the compound(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideis as follows:

Synthesis of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamide

One method for the large scale synthesis of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideis shown in Reaction Scheme I. All reactions are typically preformedunder a nitrogen atmosphere.

Step 1—Preparation of Formula (2)

The compound of formula (2) is prepared from the compound of formula (1)by reaction with hydrazine monohydrate in the absence of a solvent. Thereaction is conducted at a temperature of about 45-55° C. When thereaction is complete, the product of formula (2) is isolated by stirringwith a protic solvent in which the compound of formula (2) has limitedsolubility, for example ethanol or isopropanol. The mixture is stirredfor about 1-5 hours, and then filtered. The solid is purified bystirring with water, filtering, and washing with water followed byisopropanol and dried under vacuum, which is taken to the next stepwithout purification.

Step 2—Preparation of Formula (3)

The compound of formula (2) is then converted to a compound of formula(3) by reacting with about 1-1.2 molar equivalents of ethyl2-formyl-3-oxopropionate. The reaction is conducted in a protic solvent,preferably ethanol, at about reflux temperature, for about 2-4 hours.After cooling, to about 0° C., the solid is filtered off, washed withcold ethanol, and dried under reduced pressure. The product of formula(3) is taken to the next step without purification.

Step 3—Preparation of Final Product

The final product is prepared from the compound of formula (3) byreacting with methylamine, preferably aqueous methylamine. The reactionis carried out at about room temperature, for about 4 hours. The productof Formula I is isolated by conventional means, for example byfiltration, washing the solid with cold ethanol, and drying underreduced pressure.

Preparation of Starting Materials

(4S,2R,3R,5R)-2-(6-amino-2-chloropurin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diolis used as a starting material in step 1. This compound is commerciallyavailable.

Ethyl 2-formyl-3-oxopropanoate is used as a starting material in step 2.It is commercially available, or may be made as shown in Reaction SchemeII.

Ethyl 3,3-diethoxypropionate is reacted with ethyl formate in thepresence of a strong base, preferably sodium hydride. The reaction iscarried out at about 0-5° C., for about 24 hours. The product isisolated by conventional means, for example by the addition of water andextraction of impurities with a conventional solvent, for examplet-butylmethyl ether, acidification of the aqueous phase with, forexample, hydrochloric acid, followed by extraction with a solvent suchas dichloromethane, and removing the solvent from the dried extractunder reduced pressure. Ethyl 2-formyl-3-oxopropanoate is purified bydistillation under reduced pressure.

A preferred method for the large scale synthesis of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideis shown in Reaction Scheme III.

Step 1—Preparation of Formula (2)

As before, the compound of formula (2) is prepared from the compound offormula (1) by reaction with hydrazine monohydrate in the absence of asolvent. In this method however, a 14.3-16.7 fold molar excess ofhydrazine monohydrate is first heated to approximately 60-65° C.followed by addition of the compound of formula (1). The temperature ismaintained at approximately 60-65° C. during the reaction which takesapproximately 1 to 3 hours to complete. When the level of residualcompound of formula (1) in the mixture is not more than approximately0.10%, the reaction mixture is then cooled to approximately 40° C. Thetemperature is maintained while water is slowly added. Onceapproximately 4.2-4.9 mass equivalence have been added, the mixture iscooled to approximately 10° C. and held at that temperature for not lessthan 1 hour.

The product is then isolated by filtration, washed with water, and thenwashed with absolute ethanol. The solid is dried under vacuum at up to30° C. for not less than 12 hours and then taken to the next stepwithout further purification.

Step 2—Preparation of Formula (4)

The compound of formula (2) is then converted to a compound of formula(4) by reacting with an excess of ethyl 2-formyl-3-oxopropionate, forexample a 2-10 fold excess, preferably about 5-10 fold, ideally about6.8-7.5 fold excess. The reaction may be conducted under the sameconditions as those described for the preparation of the compound offormula (3) in Reaction Scheme I.

Alternatively, approximately 0.05 molar equivalents of an acid such asHCl may also be added to the reaction mixture. The reaction is allowedto proceed at reflux temperature, for about 2-4 hours, until the levelof residual compound of formula (2) is not more that 0.50% and theamount of any compound of formula (3) that may have formed is not morethan 2.5%. After cooling, to about 10° C., the solid is filtered off,washed with absolute ethanol, and dried under vacuum at up to 40° C. toremove residual ethanol. The product of formula (4) is taken to the nextstep without purification.

The compound of formula (4) is drawn as a (2E) alkene derivative, asthis is the major isomer formed in this reaction. However, it should benoted that a significant amount of the (2Z) alkene derivative may alsobe formed in this reaction; that is:

named as ethyl(2Z)-3-({9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)-oxolan-2-yl]-2-[4-(ethoxycarbonyl)pyrazolyl]purin-6-yl}amino)-2-formylprop-2-enoate.

Accordingly, although the compound of formula (4) is represented as the(2E) alkene derivative only, the term “compound of formula (4)” isintended to include both the instance where it is solely the (2E)isomer, and the instance where the major portion of the product is the(2E) isomer and a minor portion of the (2Z) isomer is also present. Theconversion of the compound of formula (4) to the final product byreaction with methylamine as described in Step 3 proceeds in the samemanner whether the compound of formula (4) is present as the (2E) isomeror as a mixture of the (2E) isomer and the (2Z) isomer.

Step 3—Preparation of Final Product

The final product is prepared from the compound of formula (4) byreacting with methylamine, preferably aqueous methylamine. The reactionis initially carried out at about 0-5° C. for about 8 hours, preferablyin a pressure reactor, followed by raising the temperature to 50-60° C.over about 1 hour, and maintaining the temperature for 15-30 minutes.

Alternatively, the methylamine is first placed into the pressure vesseland cooled to approximately 2.5-7.5° C. and then the compound of formula(4) is added while the temperature is maintained. The reaction proceedsuntil the level of residual compound of formula (3) is less thanapproximately 0.10%.

Upon completion, the product is isolated by conventional means, forexample by degassing under vacuum at not more than approximately 35° C.to remove excess methylamine. The vacuum is then released and themixture cooled to approximately 0-5° C. and held at the temperature for15 minutes to an hour, followed by filtration. The solid thus obtainedis washed with water followed by ethanol, and dried under reducedpressure at a temperature of no more than approximately 40° C.

This process provides(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideas its monohydrate. This polymorph can be further purified by dissolvingin dimethylsulfoxide (DMSO), filtering any solid impurities from thesolution, rinsing with additional DMSO, and precipitating themonohydrate from solution by addition to water. This method isparticularly effective when the DMSO solution and water are heated toapproximately 78-88° C. and held at that temperature with stirring forapproximately 1 hour. After stirring, the slurry is slowly cooled toapproximately 20° C. The final product is then isolated by filtration,washed with purified water followed by ethanol, and dried as describedpreviously.

EXAMPLE 1 Preparation of Ethyl-2-formyl-3-oxopropionate

A three- or four-neck round bottom flask equipped with magnetic stirbar, thermocouple, digital thermometer, gas inlet and outlet andaddition funnel was flushed with argon. Ethyl 3,3-diethoxypropionate(64.5 g) in tetrahydrofuran were charged to the addition funnel. Sodiumhydride (21.2 g of a 60% dispersion) was charged to the reaction flaskfollowed by tetrahydrofuran. The contents of the flask were cooled to0-5° C. in an ice-bath, and ethyl formate (257 g) was added. The mixturewas cooled to 0-5° C. and the contents of the addition funnel addeddropwise, maintaining an internal temperature of less than 5° C. Theice-bath was removed and the contents allowed to warm to ambienttemperature. Consumption of ethyl 3,3-diethoxypropionate was monitoredby TLC analysis. The reaction was quenched by addition of ice-water(10.6 vol), and extracted three times with methyl t-butyl ether (5.4 voleach), and the organic layers discarded. The aqueous phase was acidifiedwith conc. hydrochloric acid to a pH of 1 to 1.5. The acidified aqueouslayer was extracted three times with dichloromethane and the combinedorganic layers dried over sodium sulfate. The solvent was removed underreduced pressure, and the residue distilled under vacuum, to provideethyl 2-formyl-3-oxopropionate, 27.92 g, 70% yield.

EXAMPLE 2 A. Preparation of 2-Hydrazinoadenosine (2)

A flask equipped with a mechanical stirrer, gas inlet, gas outlet andthermocouple was flushed with argon. 2-Chloroadenosine hemihydrate (53.1g) was added, followed by hydrazine monohydrate (134 g). The mixture wasstirred while heating to 40-45° C. for 2 hours. The progress of thereaction was followed by TLC analysis. When the reaction was complete,the heat source was removed and ethanol (800 ml) was added. The mixturewas stirred for 2 hours at ambient temperature, then the precipitatecollected by filtration. The filter cake was washed with ethanol anddried under reduced pressure for 30 minutes. The solids were transferredto a clean flask equipped with a mechanical stirrer and water (300 ml)was added. The suspension was stirred at room temperature for 18 hours,and the solids isolated by filtration. The filter cake was washed withice-cold water (300 ml) followed by a wash with ice-cold ethanol (300ml). The solid was dried under reduced pressure to provide2-hydrazinoadenosine (41.38 g, 81.4% yield, 99.3% purity).

B. Alternative Preparation of 2-Hydrazinoadenosine (2)

A reaction vessel containing hydrazine hydrate (258 g, 250 ml) washeated to 40-50° C. To the warm mixture 2-chloroadenosine hemihydrate(100 g) was added in portions, maintaining the temperature between45-55° C. The temperature was kept at this temperature for two hours,and then deionized water (500 ml) was added over a period of 30 minutes,maintaining the temperature at 45-55° C. The mixture was then graduallycooled to 0-5° C. over a period of 3 hours, then stirred at thistemperature for a further 30 minutes. The solid was then filtered off,and washed with cold (2-5° C.) deionized water (200 ml), followed byethanol (400 ml). The solid was dried under vacuum for 12 hours, toprovide 2-hydrazinoadenosine.

C. Alternative Preparation of 2-Hydrazinoadenosine (2)

A reaction vessel is charged with hydrazine hydrate (1285 g). Thesolution is heated to approximately 62° C., and 2-chloroadenosine (500g) is added while maintaining the temperature of approximately 62° C.The mixture is maintained at a target of 62° C. for at least 2 hours,until the level of residual 2-chloroadenosine in the mixture is not morethan 0.10%. The mixture is cooled to approximately 40° C. and themixture is checked to verify the presence of solids. Water (2275 g) isslowly added while maintaining the temperature at approximately 40° C.The mixture is cooled to approximately 10° C. and held for not less than1 hour. The product is isolated by filtration, washed with water (1195g) and then with absolute ethanol (1885 g). The product is dried undervacuum at up to 30° C. for not less than 12 hours, to give2-hydrazinoadenosine.

EXAMPLE 3 Preparation of Ethyl1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazole-4-carboxylate(3)

Ethyl 2-formyl-3-oxopropionate (23.93 g, 0.17 mol) was placed in a flaskequipped with mechanical stirrer, gas inlet, gas outlet and refluxcondenser. 2-Propanol was added to the flask followed by2-hydrazinoadenosine (44.45 g, 0.15 mol). The mixture was heated toreflux under stirring for 2-4 hours, following the progress of thereaction by TLC analysis. When the reaction was judged complete, theheat source was removed and the mixture cooled to room temperature. Thesuspension was cooled under stirring in an ice-bath for 1.5 to 2 hours.The solids were isolated by vacuum filtration, and washed with ice-cold2-propanol. The product, ethyl1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazole-4-carboxylate,was dried under reduced pressure to a constant weight.

EXAMPLE 4 Preparation of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamide

A mixture of ethyl1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazole-4-carboxylate(46.4 g) and methylamine (40% in water, 600 ml) was stirred at ambienttemperature for about 4 hours, following the progress of the reaction byHPLC analysis. The majority of the excess methylamine was removed underreduced pressure, and the remaining mixture cooled at 0° C. for 2 hours.The solid material was filtered off, washed with ice-cold 200 proofethanol, and dried under reduced pressure, to provide(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideas its monohydrate.

The structure of the material was confirmed by ¹H NMR (see FIG. 1 andbelow). Thermal analysis (see FIG. 2) provided results consistent withthe presence of one molecule of water. X-Ray powder diffraction patternswere obtained (FIG. 3)

¹H and ¹³C NMR spectra were obtained in the following manner. Twosamples of the material obtained above were weighed out and dissolved ind₆-DMSO—5.3 mg was used for the ¹H spectra, and 20.8 mg was used for ¹³Cspectra. All spectra were acquired at ambient temperature on a JEOLEclipse⁺ 400 spectrometer operating at 400 MHz for ¹H and 100 MHz for¹³C.

Multiplicity, Label ¹³C shift(ppm) ¹H shift(ppm) splitting(Hz) 2 150.5or 150.3 — 4 156.4 — 4a 117.9 — 6 140.0 8.41 s 7a 150.5 or 150.3 — 1′86.9 5.94 D, 6.2 2′ 73.7 4.62 m 2′-OH — 5.50 D, 6.2 3′ 70.5 4.17 m 3′-OH— 5.23 D, 4.7 4′ 85.7 3.96 m 5′ 61.5 3.67, 3,57 m 5′-OH — 5.02 D, 5.7 A140.9 8.07 D, 0.8 B 120.2 — C 129.6 8.95 D, 0.8 D 161.7 — E 25.6 2.76 D,4.6 NH₂ — 7.77 br s NH — 8.35 Q, 4.6

EXAMPLE 5 A. Preparation of Ethyl(2E)-3-({9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)-oxolan-2-yl]-2-[4-(ethoxycarbonyl)pyrazolyl]purin-6-yl}amino)-2-formylprop-2-enoate(4)

A mixture of 2-hydrazinoadenosine (100 g, 0.34 mol), ethyl2-formyl-3-oxopropionate (242 g, 1.7 mol) and absolute ethanol werecharged to a reactor, and the mixture heated to reflux for 2 hours. Whenthe reaction was judged complete, the heat source was removed and themixture gradually cooled to 5-10° C. over a period of 3 hours. Theslurry was stirred for 30 minutes at this temperature, and the mixturefiltered. The solid material was washed with cold (5-10° C.) absoluteethanol, and then dried under vacuum at a temperature that did notexceed 40° C., to provide ethyl(2E)-3-({9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2-[4-(ethoxycarbonyl)-pyrazolyl]purin-6-yl}amino)-2-formylprop-2-enoate.

B. Alternative Preparation of Ethyl(2E)-3-({9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)-oxolan-2-yl]-2-[4-(ethoxycarbonyl)pyrazolyl]purin-6-yl}amino)-2-formylprop-2-enoate(4)

A reaction vessel is charged with 2-hydrazinoadenosine (450 g) andabsolute ethanol (11376 g). HCl (7.47 g) and ethyl2-formyl-3-oxopropionate (1557 g) are added. The mixture is heated toreflux and sampled until the level of residual 2-hydrazinoadenosine inthe mixture is not more than 0.50% and the level of the compound offormula (3) is not more than 2.5%. The mixture is slowly cooled toapproximately 10° C. The product, the compound of formula (4) isisolated by filtration and washed with absolute ethanol (5121 g). Theproduct is dried under vacuum at up to 40° C. until residual ethanol isnot more than 5000 ppm to give ethyl(2E)-3-({9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2-[4-(ethoxycarbonyl)-pyrazolyl]purin-6-yl}amino)-2-formylprop-2-enoate.

An elemental analysis of the product of Example 5A gave the followingresults: C, 48.75%; H, 4.86%; N, 18.05%; O, 27.57. Theoretical: C,49.72%; H, 4.74%; N, 18.45%; O, 27.09. The analysis corresponds withinexperimental error limits to the hemihydrate of the desired product. (C,48.89%; H, 4.81%; N, 18.1%; O, 28.12)

¹H and ¹³C NMR spectra were obtained in the following manner. 20.2 mg ofthe compound of formula (4) was dissolved in ˜0.75 ml of DMSO-d6, andthe spectra obtained at ambient temperature on a JEOL ECX-400 NMRspectrometer operating at 400 MHz for ¹H and 100 MHz for ¹³C. Thechemical shifts were referenced to the DMSO solvent, 2.50 ppm for ¹H and39.5 ppm for ¹³C.

Results

The ¹H and ¹³C chemical shifts are listed in Table 1. Two isomers in aratio of ˜60/30 were observed in both the ¹H and the ¹³C spectra,labeled as major and minor in the table.

¹³C Chemical ¹H Chemical Multiplicity^(b), Atom^(a) Shift (ppm) Shift(ppm) Splitting (Hz) 21(major) 192.4 9.96 d, 3.6 21(minor) 187.6 9.83 S22(minor) 167.1 — — 22(major) 165.2 — — 15(minor) 161.8 — — 15(major)161.7 — —  6(major) 153.1 — —  6(minor) 152.9 — —  2(minor) 149.4 — — 2(major) 149.3 — — 19(minor) 148.0 9.22 d, 13.0  4(minor) 147.9 — — 4(major) 147.8 — — 19(major) 147.5 9.26 d, 12.4, d, 3.6  8(major) 144.98.87 s  8(minor) 144.7 8.85 s 12 143.1 8.20-8.23 m 14(minor) 132.8 9.20d, ~0.7 14(major) 132.6 9.12 d, ~0.7  5(major) 120.7 — —  5(minor) 120.6— — 13 116.7 — — 20(minor) 107.2 — — 20(major) 106.1 — —  1′(major) 87.96.07 d, 5.3  1′(minor) 87.9 6.06 d, 5.3  4′ 85.8 4.02 q, 3.9  2′(minor)74.1 4.62 q, ~5.4  2′(major) 74.1 4.61 q, ~5.4  3′ 70.1 4.22 q, 4.2  5′61.0 3.62, 3.73 m 23, 16 60.3-60.8 4.25-4.39 m 17, 24 14.1-14.21.28-1.38 m 18(major) — 12.51 d, 12.4 18(minor) — 11.47 d, 13.0  2′-OH(major) — 5.63 d, 6.1  2′-OH (minor) — 5.62 d, 6.1  3′-OH — 5.30 d, 5.1 5′-OH — 5.08 t, 5.5The compound of formula (4) was confirmed to be a mixture of thefollowing two isomers:

EXAMPLE 6 A. Preparation of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidefrom Compound (4)

Aqueous 40% methylamine solution (1300 ml) was placed in a pressurereactor, cooled to 0-5° C., and the product of Example 5A (ethyl(2E)-3-({9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2-[4-(ethoxycarbonyl)pyrazolyl]purin-6-yl}amino)-2-formylprop-2-enoate(100 g) added. The mixture was stirred at 0-5° C. for at least 8 hours,monitoring the reaction for completion. When complete, the mixture waswarmed, maintaining the temperature between 50 and 60° C. for 1 hour,and then cooled to less than 30° C. over a period of 1 hour. When thetemperature was below 30° C., the mixture was degassed using a pressureof 100-150 mm Hg, allowing the temperature to decrease to 0-5° C. Themixture was stirred at 0-5° C. for at least 1 hour, maintaining thepressure at 100-150 mm Hg. The vacuum was then discontinued and replacedby nitrogen, maintaining the temperature at 0-5° C. for not less than 30minutes. The solid product was then filtered off, washed with water(3×500 ml), then with absolute ethanol (625 ml). The product was driedunder vacuum, not allowing the temperature to exceed 40° C., to provide(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideas its monohydrate.

B. Alternative Preparation of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidefrom Compound (4)

A pressure vessel is charged with 47% methylamine solution (10080 g).The solution is cooled and the compound of formula (4) as prepared inExample 5B above (600 g) is added while maintaining a target temperatureof 5° C. The mixture is stirred at a target temperature of 5° C. untilthe level of residual compound of formula (3) in the mixture is lessthan 0.10%. The reaction mixture is degassed under vacuum at not morethan 35° C. to remove excess methylamine. The vacuum is released and themixture is cooled to 2.5° C. and held for at least 30 min. The productis then isolated by filtration, washed with water (not less than 9000 g)and then with absolute ethanol (not less than 2964 g). The product isdried under vacuum at up to 40° C. until the residual ethanol is notmore than 5000 ppm to give crude(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideas its monohydrate.

¹H and ¹³C NMR spectra were obtained in the following manner. Twosamples of the material obtained in Example 6A were weighed out anddissolved in d₆-DMSO—5.3 mg was used for the ¹H spectra, and 20.8 mg wasused for ¹³C spectra. All spectra were acquired at ambient temperatureon a JEOL Eclipse⁺ 400 spectrometer operating at 400 MHz for ¹H and 100MHz for ¹³C.

Multiplicity, Label ¹³C shift(ppm) ¹H shift(ppm) splitting(Hz) 2 150.5or 150.3 — 4 156.4 — 4a 117.9 — 6 140.0 8.41 s 7a 150.5 or 150.3 — 1′86.9 5.94 D, 6.2 2′ 73.7 4.62 m 2′-OH — 5.50 D, 6.2 3′ 70.5 4.17 m 3′-OH— 5.23 D, 4.7 4′ 85.7 3.96 m 5′ 61.5 3.67, 3.57 m 5′-OH — 5.02 D, 5.7 A140.9 8.07 D, 0.8 B 120.2 — C 129.6 8.95 D, 0.8 D 161.7 — E 25.6 2.76 D,4.6 NH₂ — 7.77 br s NH — 8.35 Q, 4.6

An elemental analysis gave the following results: C, 43.96%; H, 4.94%;N, 27.94. Theoretical: C, 44.12%; H, 4.94%; N, 27.44%; O, 27.09. Theanalysis corresponds within experimental error limits to themonohydrate.

EXAMPLE 7 A. Purification of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidemonohydrate

A solution of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidemonohydrate, as prepared in Example 4, (100 g) in dimethylsulfoxide (300ml) was filtered through a 0.6 to 0.8 micron prefilter and a 0.2 micronfilter to remove any solid impurities. The filtrate was then slowlyadded over a period of 1 hour to deionized water (1 liter) withstirring, and the slurry thus produced stirred for not less than 1 hour.The solid was filtered off, washed with deionized water (2×1 liter), anddried under vacuum for not less than 1 hour.

The dried product was then slurried again with deionized water (1.5liter) for not less than 2 hours, filtered off, and washed withdeionized water (1 liter) followed by absolute ethanol (750 ml). Thepurified product was dried under vacuum at a temperature of not morethan 40° C. for not less than 12 hours, to provide(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidemonohydrate free of any 2-hydrazinoadenosine impurity.

B. Alternative Purification of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidemonohydrate

A solution of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidemonohydrate, (400 g) is dissolved in DMSO (1320 g), and the solution isfiltered through 0.6 to 0.8-micron and 0.2-micron in-line filters inseries. Additional DMSO (880 g) is used to rinse the filter system. Thesolution is slowly added to purified water (5000 g) while maintaining atarget temperature of 83° C. The product begins to crystallize duringthe addition, and the slurry is stirred at a target of 83° C. for about1 h. The mixture is slowly cooled to 20° C. The product is isolated byfiltration and washed with purified water (8000 g).

The solids are charged to a vessel, and purified water (6000 g) isadded. The slurry is mixed for about 1 hour. The product is isolated byfiltration and washed with purified water (4000 g) and then withabsolute ethanol (3160 g). The product is dried under vacuum at up to40° C. until the residual water content is not more than 5.5% and theresidual ethanol is not more than 2000 ppm to give(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidemonohydrate.

1. A method of synthesizing Compound A having the formula:

comprising: (a) contacting compound 1 having the formula:

with a 14.3-16.7 fold molar excess of hydrazine to provide compound 2having the formula:

(b) contacting compound 2 with an excess of ethyl2-formyl-3-oxopropionate to provide compound 4 having the formula:

(c) contacting compound 4 with methylamine.
 2. The method of claim 1,further comprising wherein the hydrazine in (a) is first heated toapproximately 60-65° C. followed by addition of compound
 1. 3. Themethod of claim 1, further comprising wherein compound 2 is isolated by:cooling the reaction mixture to approximately 40° C.; adding a 4.2-4.9mass equivalent of water while maintaining the temperature atapproximately 40° C.; and cooling the mixture to approximately 10° C.and maintaining at that temperature for at least approximately 1 hour.4. The method of claim 3, further comprising: filtering the isolatedcompound 2; washing the contents of the filter with water followed byethanol; and drying the solid that remains under vacuum at a temperaturethat does not exceed 30° C. for at least 12 hours.
 5. The method ofclaim 1, wherein the excess ethyl 2-formyl-3-oxoproionate is about a5-10 fold molar excess.
 6. The method of claim 1, wherein the excessethyl 2-formyl-3-oxoproionate is about a 6.8-7.5 fold molar excess. 7.The method of claim 1, wherein (b) further comprises adding HCl as acatalyst.
 8. The method of claim 7, wherein the HCl is added in anamount of about 0.1 equivalents or less.
 9. The method of claim 7,wherein the HCl is added in an amount of about 0.05 equivalents.
 10. Themethod of claim 1, further comprising wherein (b) is carried out atabout 80° C. and further comprises ethanol as a solvent.
 11. The methodof claim 1, further comprising wherein compound 4 is isolated by coolingthe completed reaction mixture to approximately 10° C.; filtering;washing the contents of the filter with ethanol; and drying the solidthat remains under vacuum at a temperature that does not exceed 40° C.12. The method of claim 1, further comprising wherein (c) is carried outat approximately 2.5-7.5° C.
 13. The method of claim 1, furthercomprising wherein Compound A is isolated by degassing under vacuum atnot more than 35° C. to remove excess methylamine; releasing the vacuumand cooling to 0-5° C. for approximately 15-minutes to an hour;filtering the slurry formed; washing the contents of the filter withwater followed by ethanol; and drying the solid that remains undervacuum at a temperature that does not exceed 40° C.
 14. The method ofclaim 13, further comprising wherein the Compound A is further purifiedby (i) dissolving the dried solid in a solvent; (ii) filtering any solidimpurities from the solution; (iii) rinsing with additional solvent;(iv) adding solution to purified water that is maintained atapproximately 78-88° C. thereby forming a slurry; (v) stirring theslurry; (vi) cooling the slurry; (vii) filtering; (viii) washing thecontents of the filter with water followed by ethanol; and (ix) dryingthe solid that remains under vacuum at a temperature that does notexceed 40° C.
 15. The method of claim 14, further comprising wherein thesolvent used in (i) and (iii) comprises dimethylsulfoxide.
 16. Themethod of claim 14, wherein the residual water content of the Compound Aproduct is not more than 5.5% and the residual ethanol of the Compound Aproduct is not more than 2000 ppm.